This invention was made with support provided by National Institutes of Health Grant Nos. AI22833 and AI28191. The U.S. Government retains certain rights in this invention.
This invention is directed to soluble, recombinant, fused proteins which contain a recognition site for a target molecule.
A wide variety of different molecules are present in the mammalian circulatory system, although the precise components as well as their concentration vary from time to time. These variations in the composition of the serum are in response to a spectrum of stimuli, and by sensing the changes in serum composition and concentration, the various organs of the mammals are able to respond to the stimuli. The cells of the organism recognize changes in the circulatory system by means of cell surface receptors which bind to various molecular components of the serum. It is possible to affect the way that cells of the organism will respond to the stimuli by affecting the binding of these particular components in the circulatory system to cell surface receptors.
The Complement System
One example of a system of serum components which changes in response to environmental stimuli and whose changes are sensed through binding to cell surface receptors is the complement system. The complement system is a mechanism for the recognition of foreign materials, such as microorganisms, that proceeds through two phases: the first being the covalent attachment of two complement proteins, C3 and C4, to proteins and carbohydrates that are part of the complement-activating complex. Depending on the environmental stimuli, one of two separate pathways activates an enzyme called C3-convertase which cleaves C3, releasing the C3a peptide from the alpha polypeptide of C3 and causing a major conformational change in the C3b fragment.
The second phase is the receptor-mediated binding of these complexes by various cell types, such as lymphocytes and phagocytes. In the second phase of recognition by complement, complexes containing covalently-bound fragments of C3 and C4 are bound by cells having receptors specific for these fragments. These receptors are termed complement receptors type 1 (CR1, CD35), type 2 (CR2, CD21), and type 3 (CR3, CD11b/18). The receptors are found on the surfaces of various cell types involved in immune and inflammatory responses. By modulating the response of phagocytes and lymphocytes to microorganisms and their products, this recognition program of the complement system plays a primary role in the host resistance when activation of C3 occurs through the alternative pathway. When the classical pathway has been recruited by antibody directed to the foreign molecules, binding of complement fragments plays an amplifying role.
The SCR Motif of Complement Receptor Type 1
CR1 has been extensively studied, and a structural motif of 60-70 amino acids, termed the short consensus repeat (SCR) has been found. The SCR motif is tandemly repeated 30 times in the F-allotype of CR1 and additional repeat cycles occur in other allotypes. The consensus sequence of the SCR includes 4 cysteines, a glycine and a tryptophan that are invariant among all SCR. Sixteen other positions are conserved, with the same amino acid or a conservative replacement being found in over half of the other 30 SCRs Kliekstein, et al., (1987), J. Exp. Med., (165: 1095-1112, and (1988), J. Exp. Med., 168:1699-1717; Hourcade, et al. (1988) J. Exp. Med., 168:1255-1270). The dimensions of each SCR are estimated to be approximately 2.53.0 nmxc3x972 nmxc3x972 nm.
Tandem repeats of SCRs (with the same invariant residues and similar spacing between cysteines) have been identified in 12 additional proteins of the complement system (Ahearn, et al. (1989), Adv. Immunol., 46:183-219). These proteins share a capacity for interacting with C3, C4, or C5, the set of homologous complement proteins that are subunits of the alternative and classical C3-C5 convertases and the membrane attack complex, respectively. Complement proteins containing SCRs may have activating functions (C1r, C1s, Factor B and C2), negative regulatory roles (Factor H, C4-BP, DAF, MCP, and CR1), serve as cellular receptors capable of eliciting functions of phagocytes and lymphocytes (CR1 and CR2) or promote the formation of the complement channel-forming membrane attack complex (C6 and C7). thus, the SCR is one of the most characteristic structures of the complememt system. The finding of SCRs in non-complement proteins, such as the interleukin-2 receptor alpha chain, beta-2-glycoprotein 1, and factor XIII does not necessarily indicate a complement-related function, although this possibility has not been excluded.
The first 28 SCRs from the N-terminus of CR1 may be grouped into four sequential groups, each containing seven SCRs, called long homologous repeats (LHR) and designated A, B, C, and D. LHR-D is followed by the remaining two SCRs and then by a 25 amino acid transmembrane region and a 43 amino acid cytoplasmic region that serve to anchor CR1 on the cell surface. Three complement binding sites reside in CR1: one in LHR-A specific for C4b and two additional sites in LHR-B and LHR-C specific for C3b (Klickstein, et al., 1988, supra). The two N-terminal SCRs of each LHR are involved in ligand specificity. Because complement-activating substances will bind multiple C4b and C3b molecules to their surfaces, this multivalent CR1 can interact more effectively with them than would a univalent receptor.
Other Complement Receptors
Complement receptor type 2 (CR2, CD21) is a transmembrane phosphoprotein consisting of an extracellular domain which is comprised of 15 or 16 SCRs, a 24 amino acid transmembrane region, and a 34 amino acid cytoplasmic domain (Moore, et al. (1987), Proc. Nat""l. Acad. Sci. USA, 84:9194-9198; Weis, et al. (1988), J. Exp. Med., 167:1047-1066, which are incorporated herein by reference). Electron microscopic studies of soluble recombinant CR2 have shown that, like CR1, it is an extended, highly flexible molecule with an estimated contour length of 39.6 nanometers by 3.2 nanometers, in which each SCR appears as a ringlet 2.4 nanometers in length (Moore, et al. (1989), J. Biol. Chem., 34:20576-20582).
CR2 is the B-cell receptor for both the gp350/220 envelope protein of Epstein-Barr virus (EBV) and the C3dg protein fragment of complement (Ahearn, et al., 1989, supra). An anti-CR2 monoclonal antibody (OKB7) blocks binding of both C3dg and EBV, suggesting that the natural and viral ligands bind to identical or proximal sites on the receptor (Nemerow, et al. (1985), J. Virol., 55:347-351). By means of recombinant DNA experiments with eukaryotic expression vectors expressing deletion or substitution mutants of CR2 in COS cells, the ligand binding sites of CR2 have ben localized in the two N-terminal SCRs of the molecule (Lowell, et al., (1989) J. Exp. Med., 170:1931-1946). Binding by cell-bound CR2 of the multivalent forms of C3 ligands such as iC3B and C3dg causes activation of B-cells. (Melchers, et al. (1985), Nature, 317:264-267; Bohnsack, et al. (1988), J. Immunol., 141:2569-2576; Carter, et al. (1988) J. Immunol., 457-463; and Carter, et al. (1989), J. Immunol., 143:1755-1760).
A third complement receptor, CR3, also binds iC3b. Binding of iC3b to CR3 promotes the adherence of neutrophils to complement-activating endothelial cells during inflammation (Marks, et al. (1989), Nature, 339:314). CR3 is also involved in phagocytosis, where particles coated with iC3b are engulfed by neutrophils or by macrophages (Wright, et al. (1982), J. Exp. Med., 156:1149; (1983) J. Exp. Med., 158:1338).
Soluble Complement Receptors
CR1 is a candidate for effective inhibition of complement activation. Only CR1 combines specificity for both C3b and C4b with capabilities for dissociating the C3 convertases of both pathways and for cofactor activity in the proteolytic inactivation of C3b and C4b by factor I. In addition, and probably of critical importance, these functions of CR1 are not restricted by alternative pathway activating functions, making the receptor suitable for suppressing activation by non-immunologic stimuli.
Soluble CRI (sCR1) fragments have been prepared by recombinant DNA techniques, using cDNA lacking the transmembrane and cytoplasmic domains (Fearon, et al., International Patent Application WO 89/09220, published Oct. 5, 1989), Weisman, et al., Clin. Res., 38:287A, 1990). A purified sCR1 protein produced from the vector pBSCR1c in Fearon et al., 1989 (hereafter called sCR1/pBSCR1c), bound dimeric 125I-C3b and 125I-C4b with Kds (equilibrium dissociation constant) of 1 nM and 1 nM, mediated cleavage by factor I of these proteins, and in nanomolar concentrations, inhibited classical and alternative pathway activation in human serum, indicating that its ligand building sites were intact and that it had potent in vitro inhibitory function (Weisman, et al., 1990,supra)).
In vivo complement inhibitory functions of sCR1/pBSCR12c were studied in the rat model (Weisman, et al., 1990. supra)). sCR1/pBSCR1c blocked complement activation, reduced inflammation as exemplified by decreased neutrophil accumulation in the ischemically damaged myocardium, and diminished tissue injury. Recombinant sCR1/pBSCR1c attenuates tissue damage in inflammation secondary to ischemia; it recommends itself for use in treatment of more complex autoimmune diseases known to be complement-dependent, such as immune complex-induced vasculitis, glomerulonephritis, hemolytic anemia, myasthenia gravis, rheumatoid arthritis and multiple sclerosis.
Attempts to produce a soluble CR2 analogue, have ben made (Moore, et al. (1989), J. Biol. Chem., 264:20576-20582). In analogy to the soluble CR1 system, soluble CR2 was produced in a recombinant system from an expression vector containing the entire extracellular domain of the receptor, but without the transmembrane and cytoplasmic domains. This recombinant CR2 is reported to bind to C3dg in a 1:1 complex with Kd equal to 27.5 micromolar. The binding affinity for C3dg, however, is far too low for any therapeutic application.
Soluble Receptors as Antiviral Agents
There are numerous advantages in the use of soluble viral receptors to block acute viral infection. Since variants of the virus must recognize the same cell receptor use of soluble receptors will circumvent antigenic changes and polymorphism of viral envelope proteins or strain variation. Further, the viral-binding domain(s) of cellular receptors would not likely be antigenic, toxic, or immunosuppressive. (Nemerow, et al. (1990) J. Virol., 64:1348-1352). Because the soluble receptor is not used as an immunogen, effectiveness would not require the use of adjuvants, and furthermore, efficacy would not be dependent on the presence of an intact immune system.
CR2 is one of the primary determinants of Epstein-Barr virus tropism because it specifically binds virions to the cell membrane. Soluble CR2 was produced in a recombinant system from an expression vector containing the entire extracellular domain of the receptor, but without the transmembrane and cytoplasmic domains. This recombinant CR2 is reported to bind to the Epstein-Barr proteins gp350/220 in a 1:1 complex with Kd=3.2 nM. (Moore, et al. (1989), J. Biol. Chem., 264:20576-20582).
The attempt to block viral binding by administering a soluble form of the membrane-bound receptor protein for a virus has been explored in other viral systems, particularly in AIDS. The AIDS virus receptor protein, CD4, was prepared in soluble form by recombinant methods, using DNA encoding the extracellular domain but not the transmembrane region or the cytoplasmic region (Hussey, et al. (1988), Nature, 331:78-81). This recombinant protein was successful in blocking AIDS infection of cultured cells, but when injected into patients, the recombinant protein was rapidly cleared, with the half-life of the major phase of drug elimination being approximately 1 hour (Kahn, et al. (1990), Ann, Intern. Med., 112:254-261).
Hybrid Immunoglobulin Proteins
In order to overcome the short-half life of soluble CD4, hybrid molecules were prepared by recombinant DNA technology in which DNA encoding the binding region of CD4 was substituted for DNA encoding the variable region of a murine immunoglobulin molecule (Capon, et al. (1989), Nature, 337:525-531 and WO89/029922). This is possible because CD4 is itself part of the immunoglobulin gene superfamily, and therefore it has a homologous structure to the variable region that was replaced. The CD4 hybrid based on murine immunoglobulin showed a substantial increase in the serum half-life in rabbits compared to soluble CD4.
Hybrid antibodies were also produced by Bruggemann, et al. (1987, J. Exp. Med., 166:1351-1360), to study the effect of changes in various parts of the molecule on the function of antibodies where the antigen binding region is held constant. Here again, the structure of the peptide sequence inserted into the immunoglobulin molecule was similar to that of the peptide which it replaced. The study showed that regions of the immunoglobulin structure can be substituted with homologous structures without disrupting the structure of the whole molecule.
In order to obtain enough T-cell receptor protein for biochemical studies, Gascoigne, et al. (1987), Proc. Natl. Acad. Sci. USA, 84:2936-2940) constructed an expression vector encoding a hybrid T-cell receptor-immunoglobulin protein. The T-cell receptor is a member of the immunoglobulin gene superfamily, encoded by a genomic DNA composed of a number of similar gene segments which rearrange to form the final coding sequence for the receptor protein. The final rearranged sequence has variable, diversity and joining segments in parallel with immunoglobulin genes. The hybrid receptor protein was constructed by replacing the variable region of an expression vector for a heavy immunoglobulin chain with the rearranged variable region of a T-cell receptor. This vector was then expressed in a cell line which only secreted the light chain. The transformed cell line secreted a chimeric protein which had both immunoglobulin and T-cell receptor determinants.
The above prior art substituted only DNA sequences encoding peptide domains which have similar homology units to immunoglobulin peptides derived from the immunoglobulin gene superfamily (Hood, et al. (1985), Cell, 40:225-229). The DNA sequences corresponding to a homology unit(s) of the immunoglobulin chain were removed and replaced by DNA encoding a similar homology unit from another protein of the immunoglobulin supergene family without disrupting the ability of a host cell to express the hybrid protein.
It is one object of this invention to provide a soluble protein capable of specifically binding a target molecule, said protein having a good half-life of clearance from the mammalian circulatory system.
It is another object of this invention to provide a soluble protein capable of specifically binding the target molecule, said protein having an enhanced affinity for the target molecule.
It is a further object of this invention to provide a soluble construct capable of specific multivalent binding to complement proteins.
It is still another object of this invention to provide a soluble construct with a compact structure which will be better able to diffuse into tissues from intravascular space.
It is yet another object of this invention to provide a soluble construct which will compete with the cell-bound receptors and which will persist in the mammalian circulatory system.
It is a further object of this invention to provide a method for inhibiting complement-dependent cellular activation in animals by administration of a soluble construct which is stable in the circulatory system.
It is yet another object of this invention to provide a method for inhibiting complement activation in animals by administering a soluble construct which is stable in the circulatory system.
In one of its aspects, this invention contemplates a soluble recombinant fused protein which is stable in the mammalian circulatory system comprising a polypeptide which contains a recognition site for a target molecule and is joined to the N-terminal end of an immunoglobulin chain. The use of the recombinant fused protein in therapy is also contemplated.
In a related aspect, this invention contemplates an expression vector and a method for producing an expression vector encoding the recombinant fused protein which comprises modifying an expression vector for an immunoglobulin chain by inserting a DNA sequence encoding a binding or recognition site between the DNA sequence encoding the leader peptide and the DNA sequence encoding the N-terminal end of an immunoglobulin chain. A host cell is also contemplated which contains the vector, the cell preferably expressing a complementary immunoglobulin chain, so that a complete immunoglobulin molecule or fragment is secreted which carries the binding or recognition site fused to the N-terminus of at least one immunoglobulin chain.
The recombinant fused protein of this aspect of the invention is soluble and will be relatively stable in aqueous medium, particularly he mammalian circulatory system, because of the stability and solubility of the immunoglobulin molecule. Where the recombinant fused protein is secreted as part of an antibody molecule, said molecule possesses polypeptide recognition moieties attached to the N-terminal ends of at least two of the four immunoglobulin chains present and also possesses the enhanced binding affinity for the target molecule which multivalency confers. The flexibility inherent in the immunoglobulin structure, particularly due to the hinge portion of the antibody molecule, permits movement of the polypeptide binding moieties relative to each other to facilitate adaptation of the three-dimensional arrangement of binding sites to the three-dimensional arrangement of complementary sites on the target.
While the use of a recombinant fused immunoglobulin protein containing multiple short consensus repeats having a complement binding site is a preferred embodiment of the invention, the invention also more broadly contemplates constructs comprising a plurality of peptides containing short consensus repeats having a complement binding site attached to a soluble, physiologically compatible carrier and the use of such constructs in therapy.
Such constructs provide significant benefits with respect to enhancing binding affinity by presenting multiple binding sites (multi-valency). The enhanced affinity of the constructs of this invention provides important benefits when the constructs are used in therapy. Such benefits are particularly important for therapy employing short chain repeats derived from CR2 since mature CR2 contains only a single binding site with low affinity.